Differing requirements for the Arabidopsis Rad51 paralogs in meiosis and DNA repair

Differing requirements for the Arabidopsis Rad51 paralogs inmeiosis and DNA repair

Jean-Yves Bleuyard, Maria E. Gallego, Florence Savigny and Charles I. White*

CNRS UMR6547, Universite Blaise Pascal, 24, avenue des Landais, 63177 Aubiere, France

Received 8 October 2004; revised 12 November 2004; accepted 16 November 2004.*For correspondence (fax !33 473 407 777; e-mail [email protected]).

Summary

In addition to the recombinase Rad51, vertebrates have five paralogs of Rad51, all members of the Rad51-

dependent recombination pathway. These paralogs form two complexes (Rad51C/Xrcc3 and Rad51B/C/D/

Xrcc2), which play roles in somatic recombination, DNA repair and chromosome stability. However, little is

known of their possible involvement in meiosis, due to the inviability of the corresponding knockout mice. We

have recently reported that the Arabidopsis homolog of one of these Rad51 paralogs (AtXrcc3) is involved in

DNA repair and meiotic recombination and present here Arabidopsis lines carrying mutations in three other

Rad51 paralogs (AtRad51B, AtRad51C and AtXrcc2). Disruption of any one of these paralogs confers

hypersensitivity to the DNA cross-linking agentMitomycin C, but not to c-irradiation. Moreover, the atrad51c-1

mutant is the only one of these to show meiotic defects similar to those of the atxrcc3 mutant, and thus only

the Rad51C/Xrcc3 complex is required to achieve meiosis. These results support conservation of functions of

the Rad51 paralogs between vertebrates and plants and differing requirements for the Rad51 paralogs in

meiosis and DNA repair.

Keywords: DNA repair, meiosis, recombination, RAD51B, RAD51C, XRCC2.

Introduction

Homologous recombination (HR) and non-homologousend-joining (NHEJ) are the two major pathways for DNAdouble-strand break (DSB) repair (reviews by Dudas andChovanec, 2004; Lees-Miller and Meek, 2003). While NHEJ isan error-prone pathway, frequently leading to deletions and/or insertions, repair of DNA DSBs through HR generallypreserves the integrity of the genomic material. In addition,HR events play an essential role in assuring proper meioticchromosomal disjunction and represent an essential mech-anism to create genetic diversity. The proteins involved inHR mechanisms in eukaryotic cells have been extensivelystudied and mainly belong to the RAD52 epistasis group,first identified in budding yeast (for review see Dudas andChovanec, 2004). Within this group, the sub-family of theRad51-like proteins has been the object of a considerableinterest as the finding that Rad51 is the homolog of thebacterial recombinase RecA (Aboussekhra et al., 1992;Shinohara et al., 1992). In addition to the highly conservedrecombinase Rad51, three Rad51-like proteins have beenidentified in budding yeast: Dmc1 is a meiosis-specificRad51-like protein and is conserved in many eukaryotes,

while Rad55 and Rad57 are expressed ubiquitously andseem to be specific to yeasts. All these Rad51-like proteinsplay roles in DSBs repair and/or HR (for review see Dudasand Chovanec, 2004).

As well as Rad51 and Dmc1, the genomes of vertebratesencode five additional Rad51-like proteins, referred to as theRad51 paralogs: Rad51B (Albala et al., 1997; Cartwrightet al., 1998b; Rice et al., 1997), Rad51C (Dosanjh et al.,1998), Rad51D (Cartwright et al., 1998b; Pittman et al.,1998), Xrcc2 (Cartwright et al., 1998a; Liu et al., 1998) andXrcc3 (Liu et al., 1998; Tebbs et al., 1995). Two-hybrid andimmunoprecipitation experiments have shown that thesefive Rad51 paralogs form two complexes: an heterodimercomposed of Rad51C and Xrcc3 (CX3) and an heterotetr-amer composed of Rad51B, Rad51C, Rad51D and Xrcc2(BCDX2) (Liu et al., 2002; Masson et al., 2001; Miller et al.,2002, 2004; Schild et al., 2000; Wiese et al., 2002). Theembryonic lethality observed in individual knockouts ofmouse RAD51B, RAD51D and XRCC2 shows that, as is thecase for Rad51, the Rad51 paralogs are required for animalsviability (Deans et al., 2000; Pittman and Schimenti, 2000;

ª 2004 Blackwell Publishing Ltd 533

The Plant Journal (2005) 41, 533–545 doi: 10.1111/j.1365-313X.2004.02318.x

Shu et al., 1999; Tsuzuki et al., 1996). However, in contrast toRad51, knockouts of the Rad51 paralogs in chicken DT-40cells do not lead to cell death, indicating that these proteinsare not required for vertebrate cells viability (Sonoda et al.,1998; Takata et al., 2000, 2001). The numerous experimentsperformed with mutant cells defective in the different Rad51paralogs have shown that they all play roles in somaticrecombination, DNA repair and chromosome stability(Brenneman et al., 2000; Cui et al., 1999; Deans et al., 2000,2003; French et al., 2002; Godthelp et al., 2002; Johnsonet al., 1999; Liu et al., 1998; Mohindra et al., 2004; Pierceet al., 1999; Takata et al., 2000, 2001; Tebbs et al., 1995).However, the embryonic lethality ofmutants defective in anyone of the Rad51 paralogs has complicated investigation oftheir possible involvement in meiosis (Deans et al., 2000;Pittman and Schimenti, 2000; Shu et al., 1999).

The absence of Rad51 foci following DNA damagingtreatment in cells defective in any one of the Rad51 paralogssuggests a role in the initial steps of HR process (Bishopet al., 1998; Godthelp et al., 2002; Liu, 2002; O’Regan et al.,2001; Takata et al., 2000, 2001; Tarsounas et al., 2004). Thishypothesis is supported by several biochemical studies. Invitro Rad51-dependent strand exchange reactions haveshown that a sub-complex composed of Rad51B and Rad51Cfacilitates the assembly of the Rad51-ssDNA nucleoproteinfilament in the presence of RPA and thus has a mediator rolesimilar to that of the Rad55-Rad57 heterodimer in yeast (Lioet al., 2003; Sigurdsson et al., 2001; Sung, 1997). TheRad51C protein, the CX3 complex and a Rad51D/Xrcc2 sub-complex possess homologous pairing activities similar tothat of Rad51 (Kurumizaka et al., 2001, 2002; Lio et al., 2003).

Several recent studies however support a later role for theRad51 paralogs. In the absence of the Xrcc3 protein, bothconversion tract lengths and the frequency of discontinuoustracts are increased (Brenneman et al., 2002). In vitro, theRad51B protein and BCDX2 complex preferentially bindbranched DNA strands, such as Holliday junctions (HJ)(Yokoyama et al., 2003, 2004), and Rad51C and Xrcc3 playroles in HJ resolution (Liu et al., 2004). Finally, the meioticdefects observed in an Arabidopsis xrcc3 mutant suggestthat the Xrcc3 protein plays a post-synaptic role (Bleuyardand White, 2004).

The genome of Arabidopsis thaliana codes for sevenRad51-like proteins. In addition to the previously identified

Rad51, Dmc1, Rad51C and Xrcc3 Arabidopsis homologs(Doutriaux et al., 1998; Klimyuk and Jones, 1997; Osakabeet al., 2002; Sato et al., 1995; Urban et al., 1996), theconstruction of a phylogenetic tree has allowed us to identifythe Arabidopsis homologs of Rad51B, Rad51D and Xrcc2,showing that Arabidopsis has the same family of Rad51-likeproteins as vertebrates. To investigate the conservation offunctions of the Rad51 paralogs between vertebrates andplants, we identified mutants defective for three of the fourother Arabidopsis Rad51 paralogs. Absence of any one ofthe Rad51B (AtRad51B), Rad51C (AtRad51C) and Xrcc2(AtXrcc2) Arabidopsis homologs confers hypersensitivityto the DNA cross-linking agent Mitomycin C (MMC), but notto ionizing radiation. Furthermore, only mutants impairedfor theAtRAD51C gene showmeiotic defects similar to thoseof atxrcc3 mutants. These results clearly show that the roleof the Rad51 paralogs in DNA repair is conserved betweenvertebrates and plants and that only AtRad51C and AtXrcc3(Bleuyard and White, 2004), which together form the CX3complex, play essential roles in meiosis in Arabidopsis, andvery probably in other higher eukaryotes.

Results

Identification and molecular characterization of Arabidopsismutants defective for the Rad51 paralogs

The genome of the model plant A. thaliana codes for sevenproteins of the Rad51 recombinase family. Previous studieshave reported the identificationof Rad51 (AT5G20850), Dmc1(AT3G22880), Rad51C (AT2G45280) and Xrcc3 (AT5G57450)homologs (Doutriaux et al., 1998; Klimyuk and Jones, 1997;Osakabe et al., 2002; Sato et al., 1995; Urban et al., 1996). Todefine the relationships existing between ArabidopsisRad51-likeproteinsandRad51-likeproteins fromothermodelspecies, we performed a phylogenetic analysis with 23Rad51-like proteins from Arabidopsis, Drosophila, humanand Saccharomyces (Figure 1a). The resulting phylogenetictree clearly shows that Arabidopsis has a single homolog foreach of the five Rad51 paralogs first identified in vertebrates(AT2G28560, AT1G07745 and AT5G64520 products corres-pond respectively to the AtRad51B, AtRad51D and AtXrcc2proteins). The A-type nucleotide binding consensus aminoacid sequence [G/A]XXXXGK[S/T] (Walker A motif) is

Figure 1. Arabidopsis genome encodes homologs of the five Rad51 paralogs.(a) The dendrogram illustrates the sequence relationships among 23 Rad51-like proteins in Saccharomyces cerevisiae (Sc), Drosophila melanogaster (Dm), Human(Hs) and Arabidopsis (At). The branch lengths are proportional to the sequence divergence. Numbers along branches are bootstrap values (1000 replicates)calculated using the PHYLIP package. The scale represents 0.1 substitutions per site.(b) The core-conserved sequences were aligned using the ClustalX program. The amino acid sequences of the proteins are shown in the single-letter code. Gaps areindicated by dashes. Conserved amino acids are black shaded and similar amino acids are gray shaded. The positions of the amino acids in each protein are shownat left and right. Black boxes indicate the positions of Walker motifs A and B.Accession numbers for 23 deduced amino acids sequences used in this analysis are as follows: AtDmc1 (AAC49617), AtRad51 (CAA04529), AtRad51B (NP_180423),AtRad51Ca (BAB64343), AtRad51D (NP_172254), AtXrcc2 (NP_851268), AtXrcc3 (BAB64342), DmCG2412 (NP_610466), DmCG6318 (NP_573302), DmRad51(BAA04580), DmSpn-B (NP_476740), DmSpn-D (AAP13056), HsDmc1(BAA10970), HsRad51 (BAA02962), HsRad51B (AAC39723), HsRad51C (AAC39604), HsRad51D(AAC39719), HsXrcc2 (CAA70065), HsXrcc3 (AAC05368), ScDmc1 (AAA34571), ScRad51 (BAA00913), ScRad55 (AAA19688), ScRad57 (AAA34950).

534 Jean-Yves Bleuyard et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 533–545

(a)

(b) Walker A motif(G/A)XXXXGK(S/T)

Roles of Arabidopsis Rad51 paralogs 535


conserved in the Arabidopsis Rad51 paralogs, except forAtXrcc2, while the B-type binding consensus amino acidssequence hhhhD (Walker B motif) is conserved in the fiveArabidopsis Rad51 paralogs (Figure 1b) (Higgins et al., 1985;Walker et al., 1982).

In order to investigate the roles of the ArabidopsisRad51 paralogs in meiosis and the cellular responses toDNA damage, we searched for mutants in public T-DNAinsertion line collections. Three lines carrying T-DNAinsertions in the ATRAD51B (Salk_024755), ATRAD51C(Salk_021960) and ATXRCC2 (Salk_029106) codingsequences were found in the SIGnAL T-DNA expressdatabase (Alonso et al., 2003) and we have named thecorresponding alleles atrad51b-1, atrad51c-1 and atxrcc2-1respectively. Plants homozygous for atrad51b-1, atrad51c-1and atxrcc2-1 T-DNA insertions were identified by PCR inthe T3 seeds provided by the Nottingham Arabidopsis

Stock Centre. A sterility phenotype was observed inplants homozygous for atrad51c-1, thus the atrad51c-1T-DNA insertion was kept at the heterozygous state andatrad51c-1 mutant plants were identified by PCR in theprogeny of ATRAD51C-1!/) plants.

To characterize the insertions molecularly, the T-DNAjunctions were amplified and the PCR products sequenced(Figure 2). The atrad51b-1 allele T-DNA is inserted in intron 4and is associated with a deletion of 14 bp, including the first4 bp of exon 5 (Figure 2a). The atrad51c-1 allele T-DNA isinserted in exon 3 and is associated with a deletion of 47 bp,containing the end of exon 3 and the beginning of intron 3(Figure 2b). The atxrcc2-1 allele T-DNA is inserted in intron 5,3 bp after the end of exon 5, and is associatedwith a deletionof 1 bp (Figure 2c). These three T-DNA insertions aresurrounded by two left borders in opposite orientations,designated as LB1 and LB2 (Figure 2 diagrams). In the case

(a)

! 14 b

LB1 LB2T-DNA(+949) (+964)

Filler DNA 70 bAtRAD51B

ATG (+1) STOP (+2244)

5"-UTR 3"-UTR

500 bo546 o548 o547 o549

TTCTTTC aactta...tcc aag...tactgc CAAGAAC

T-DNALB1 LB2AtRAD51B AtRAD51B

+945 +965

Filler DNA

{

o548/o549o546/o547

Control

Wild Type atrad51b-1{(b)

LB1 LB2T-DNA

! 47 bATG (+1)

STOP(+1553)

STOP (+1939)

(+688) (+734)

AtRAD51C

A(n)

A(n)

500 b

#3"-UTR5"-UTR

$

o450 o453o554 o454

+685

AGGGATGT aaacaa...aagcgt CAATTTGAGAT

T-DNALB1 LB2AtRAD51C AtRAD51C

+740{

o453/o454

o450/o554

Control

Wild Type atrad51c-1{(c)

AATCGGTTA aacaaa...gtcaat TTGGTTAAT

T-DNALB1 LB2AtXRCC2 AtXRCC2

+1495 +1505{

o552/o553

o550/o551

Control

Wild Type {atxrcc2-1

LB1 LB2T-DNA

! 1 bATG (+1)

21

STOP (+2174)

STOP (+2136)

(+1499) (+1501)

AtXRCC2

3"-UTR

500 bo550 o552o551 o553

Figure 2. Molecular characterization of Arabidopsis Rad51 paralog T-DNA insertion mutants.The diagrams show the genomic structure of ATRAD51B (a), ATRAD51C (b) and ATXRCC2 (c) loci, with the sites of T-DNA integrations. The gray boxes represent theexons and the white boxes indicate 5¢ and 3¢ UTRs. The position of the T-DNA insertions is indicated by triangles. Sequences of the T-DNA junctions and RT-PCRdetection of theATRAD51B (a),ATRAD51C (b) andATXRCC2 (c) transcripts are presented at the right of each panel. Thewhite box indicates the T-DNA insertion. LB1and LB2 indicate the two left borders surrounding the T-DNA insertions, and arrows indicate their respective orientation. Micro-homologies between the T-DNAends and the genomic sequence are indicated in gray. The positions of the primers used for the RT-PCR detection of ATRAD51B (a), ATRAD51C (b) and ATXRCC2 (c)transcripts are represented on the diagrams. Amplification of the adenosin phosphoribosyl transferase (APT1) transcript has been used as a control for reversetranscription. The numbers represent positions relative to the start codon.



of atrad51b-1, sequencing showed that the T-DNA insertionis followed by an insertion of 70 bp of filler DNA (Figure 2b).

Plants homozygous for the T-DNA insertions were selec-ted by PCR, and semiquantitative RT-PCR analysis per-formed to assess the presence of ATRAD51B, ATRAD51Cand ATXRCC2 transcripts in total RNA isolated from wildtype and mutant flower buds. ATRAD51B, ATRAD51C andATXRCC2 transcripts were detected in the wild type. Incontrast, the ATRAD51C mRNA was not detectable inatrad51C-1 plants, and only truncated ATRAD51B andATXRCC2 mRNAs were detected in atrad51b-1 and atxrcc2-1 plants respectively (Figure 2). The T-DNA insertions in theatrad51b-1, atrad51c-1 and atxrcc2-1 mutants thus preventthe production of the full-length mRNAs of ATRAD51B,ATRAD51C and ATXRCC2 respectively. Furthermore, thecomplete absence of ATRAD51C transcript in atrad51c-1plants shows that it is a null allele, while atrad51b-1 andatxrcc2-1 may potentially encode truncated proteins.

atrad51b-1, atrad51c-1 and atxrcc2-1 mutant plants arehypersensitive to Mitomycin C, but not to c-irradiation

Studies performed with Chinese Hamster Ovary and DT40(Chicken B-Lymphocyte) cell lines have shown that muta-tions of the different Rad51 paralogs confer moderate sen-sitivity to DNA DSB inducing agents such as c-rays andhypersensitivity to DNA cross-linking agents such as MMC(Godthelp et al., 2002; Liu et al., 1998; Takata et al., 2001).Our recent work with an Arabidopsis atxrcc3mutant showedthat plants lacking the AtXrcc3 protein were slightly sensi-tive to bleomycin, a c-ray mimetic agent, and much moresensitive to MMC, suggesting a conservation of the role ofArabidopsis Rad51 paralogs in response to DNA damage(Bleuyard and White, 2004).

To confirm the involvement of AtRad51B, AtRad51C andAtXrcc2 proteins in the cellular response to DNA damage,seeds from wild type, atrad51b-1 and atxrcc2-1 plants andself-fertilized heterozygous ATRAD51C-1!/) plants wereeither irradiated with c-rays and sown on germinationmedium, or sown on plates containing germinationmediumand increasing doses of MMC. After 2 weeks, plants werescored for c-irradiation or MMC sensitivity. In the absence oftreatment, most plants developed at least four true leaves (inaddition to the cotyledons), thus plants with three trueleaves or less were considered to be sensitive to c-rays orMMC. Previous studies on DNA repair defective mutantshave shown that a dose of 100 Grays (Gy) allow discrimin-ation between atku80, atlig4 and atatm radiosensitivemutants and wild-type plants (Friesner and Britt, 2003;Garcia et al., 2003). In our experiments, no significantdifference was found between wild type and atrad51b-1,atrad51c-1 and atxrcc2-1 mutant plants, even at a dose of200 Gy (data not shown). Similar c-irradiation assays wereperformed on the seedlings of an ATXRCC3!/) plant and, as

for atrad51b-1, atrad51c-1 and atxrcc2-1 mutant plants, nosignificant difference was found when compared with thewild type (unpublished data).

Examples of non-sensitive and sensitive plants and dose–response curves for the percentage of sensitive plants areshown in Figure 3. atrad51b-1 and atxrcc2-1 mutant plantsclearly show hypersensitivity to MMC. Due to the sterility ofthe atrad51c-1 mutant plants, the MMC hypersensitivityof atrad51c-1 plants was assayed on the progeny ofATRAD51C-1!/) plants, one quarter of which are mutants(Figure 3c). That the sensitive plants were the atrad51c-1mutants was verified by PCR genotyping in one experimentand all 27 sensitive plants scored were mutants. TheAtRad51B, AtRad51C and AtXrcc2 proteins are thus requiredto repair DNA cross-links but are not essential for the repairof DSBs, presumably due to the repair of DSBs by NHEJ.

atrad51c-1 mutants, but not atrad51b-1 or atxrcc2-1, aresterile

In the progeny of self-fertilized heterozygousATRAD51C-1!/)

plants (41 plants screened): 10 (24.4%) were sterile and 31(75.6%) were fertile, corresponding well to the 3:1 segrega-tion expected for a single Mendelian locus (chi-squared, 1d.f. " 0.008). Genotyping confirmed that the sterile plantswere exclusively homozygous atrad51c-1mutants. atrad51c-1 plants produce atrophied siliques, which are devoid of anyseed, while heterozygotes for atrad51c-1 and homozygotesfor atrad51b-1 or atxrcc2-1 do not show any fertility defectsand all mutant plants grew normally with normal vegetativedevelopment (data not shown).

We investigated the origin of the sterility of atrad51c-1plants and confirmed the absence of such defects inatrad51b-1 and atxrcc2-1 mutant plants (Figure 4). Antherswere dissected from wild type and atrad51b-1, atrad51c-1and atxrcc2-1 mutant flower buds and stained as describedby Alexander (1969) to assess pollen grain viability (Fig-ure 4a–d). While none of the observed atrad51c-1 antherscontained any viable (red-purple) pollen grains, anthersfrom atrad51b-1 and atxrcc2-1 mutant plants could not bedifferentiated from those of the wild type in terms of thenumber of viable pollen grains produced. To assess femalegametophytic defects, we monitored post-meiotic nucleardivisions during embryo sac development. In thewild type, asingle megaspore mother cell differentiates in each ovuleand undergoes meiosis (Figure 4e). Meiosis in femaletissues is followed by degeneration of three of the fourmeiotic products, to preserve a single functional megaspore(Figure 4f). The functional megaspore nucleus then under-goes three divisions to produce the eight-nuclei embryo sac,which is the mature female gametophyte (Figure 4g,h). Inatrad51c-1 ovules, the megaspore mother cell could not bedifferentiated from the wild type (Figure 4i), but gameto-phytic development is blocked after meiosis. The presence



of a single degenerative cell, which persists throughoutembryo sac development, suggests that the megasporemother cell is unable to properly achieve meiosis inatrad51c-1 ovules (Figure 4j–l). In some cases, one of themeiotic products is preserved, but such products were neverable to proceed further than the first post-meiotic division(data not shown). The AtRad51C protein is thus required tocomplete both male and female gametogenesis, as is thecase for AtXrcc3. In contrast, AtRad51B and AtXrcc2 are notrequired to achieve gametogenesis.

The AtRad51C protein is required to ensure chromosomestability during meiosis

Meiotic progression in wild type, atrad51b-1, atrad51c-1 andatxrcc2-1 pollen mother cells (PMCs) was examined byfluorescence microscopy after DAPI staining of chromo-somes. In the wild type, the 10 Arabidopsis chromosomescondense during meiotic prophase I (Figure 5a) and can beseen as five bivalents (corresponding to paired homologouschromosomes) in metaphase I (Figure 5b). Homologouschromosomes then separate from each other andmigrate to

the opposite poles of the cell in anaphase I (Figure 5c). Thesecond meiotic division starts with the alignment of chro-mosomes in metaphase II (Figure 5d), followed by separ-ation of sister chromatids in anaphase II (Figure 5e).Telophase II ensues, chromosomes decondense (Figure 5f)and cytoplasm is partitioned to produce a tetrad containingfour haploid microspores, which will differentiate intomature pollen grains.

In atrad51c-1mutant PMCs, prophase I proceeds normallyup to pachytene (Figure 6a), but in place of five expectedbivalents, a varying number of entangled chromosomefragments can be seen in metaphase I figures (Figure 6b,c).This chromosome fragmentation becomesmore apparent inanaphase I, with random segregation of chromosomefragments and the presence of bridges, indicating chromo-some fusion events and the presence of dicentric chromo-somes (Figure 6d,e). In metaphase II, most of the visiblechromosome fragments are aligned on the spindle,with some fragments scattered throughout the cytoplasm(Figure 6f,g). Anaphase II separates several groups ofchromosome fragments (Figure 6h,i) and is followed bychromosome decondensation in telophase II (Figure 6j).

}W

ild T

ype

atxr

cc2-

1at

rad5

1b-1

(a)at

xrad

51c-

1

Mitomycin C concentration

% o

f sen

sitiv

e pl

ants

0 µM 10 µM 20 µM 40 µM

0

5

10

15

20

25

30Wild TypeAtRad51C-1+/-(c)

(b)

Mitomycin C concentration

% o

f sen

sitiv

e pl

ants

Wild Typeatrad51b-1atxrcc2-1

10

20

30

40

50

60

70

80

90

0 µM 10 µM 20 µM 40 µM

0

Figure 3. Mutations in Arabidopsis Rad51 paralogs confer hypersensitivity to the DNA cross-linking agent, Mitomycin C (MMC).(a) Comparison of non-sensitive and sensitive plants at 40 lm MMC. In the absence of MMC, all plants develop at least four true leaves (excluding the cotyledons),thus plants with three leaves or less were considered as sensitive. Scale bars " 1 cm.(b, c) The percentage of sensitive plants was used to produce anMMC dose–response curve. Values represent three replicates, each replicate containing an averageof 100 plants per dose. Error bars are # 1 standard deviation.



The observation of bridges in anaphase II figures suggeststhat fused chromosome fragments are still present at thisstage and that chromosome fragmentation continues to theend of anaphase II. Meiosis in atrad51c-1 PMCs finally givesrise to ‘polyads’, containing variable numbers of productswith variable DNA contents. In contrast, meiotic progressionin the atrad51b-1 and atxrcc2-1 mutants is normal. Thesetwo Rad51 paralogs are thus not required to ensurechromosome stability during meiosis (data not shown).Taken together, these results indicate that, as previouslyshown for AtXrcc3, the AtRad51C protein is required toachieve meiosis. The chromosome fragmentation observedin atrad51c-1 meiosis presumably resulting from mis- orun-repaired meiotic double-strand breaks, in agreementwith a role of AtRad51C in HJ resolution (Liu et al., 2004;Symington and Holloman, 2004).

Discussion

Wereport here the identificationand characterizationof threeArabidopsis mutants, defective for the Rad51 paralogs

AtRad51B, AtRad51C and AtXrcc2 respectively. The firststriking result from this study is that, in contrast to verte-brates, mutations in any one of these Arabidopsis Rad51paralogs do not impair plant viability (Deans et al., 2000;Pittman and Schimenti, 2000; Shu et al., 1999). Studies car-ried out with vertebrate cell lines defective for the Rad51paralogs have shown that these proteins are involved in DNArepair, with mutant cell lines showing relatively moderatesensitivity to DSB inducing agents such as c-rays and highsensitivity to chemicals inducing the formation of interstrandcross-links (ICLs) (Godthelp et al., 2002; Liu et al., 1998;Takata et al., 2000, 2001). However, Drosophila xrcc3 (spn-B)and rad51c (spn-D) mutants do not present DNA repairdefects and this role of the Rad51 paralogs is thus not self-evident (Abdu et al., 2003). In previous work, we have shownthat cultured cells defective for the Arabidopsis XRCC3homologue have increased sensitivity to the radiomimeticagent Bleomycin (Bleuyard andWhite, 2004). Here we reportthat mutations in either ATRAD51B, ATRAD51C or ATXRCC2genes did not increase sensitivity of plants to c-rays. Similarresults were also obtained with ATXRCC3-defective plants

(d)(c)(b)(a)

(g) (h)(f)(e)

(l)(k)(j)(i)

Figure 4. AtRad51C, but not AtRad51B and AtXrcc2, is required for gametophytic development.(a–d) Alexander staining was applied to anthers fromwild type (a), atrad51b-1 (b), atrad51c-1 (c) and atxrcc2-1 (d) plants to discriminate viable (stained in red-purple)and dead (stained in green) pollen grains.Wild type (e–h) and atrad51c-1 (i, j) ovules were cleared to observed embryo sac development. (e, i) megaspore mother cell; (f) functional megaspore; (g) two-nucleistage; (h) four-nuclei stage; (j–l) degenerative cell observed at different developmental stages.



(unpublished data), and we ascribe this difference to the dif-ferent effects of chronic exposure of cultured cells to Bleo-mycin compared with the DNA breakage produced by theacute c-irradiation. Our data thus indicate that mutations infour different Arabidopsis Rad51 paralogs confer little or nosensitivity to DSB-inducing agents. In contrast, the hyper-sensitivity to MMC observed in atrad51b-1, atrad51c-1 andatrad51-1 mutant plants confirms the important role of Ara-bidopsis Rad51 paralogs in the repair of ICLs. Taken together,our data strongly support functional conservation of theRad51paralogs inDNArepairbetweenvertebratesandplants(this study; Bleuyard and White, 2004).

Meiotic function of the CX3 complex

Yeast rad51 mutants are unable to repair meiosis-specificDSBs and their ability to produce viable spore is dramaticallyreduced (Cao et al., 1990; Shinohara et al., 1992). Similarly,Drosophila and Caenorhabditis mutants defective for Rad51show defects in chromosomemorphology during oogenesis

and thus, reduced fertility (Rinaldo et al., 2002; Staeva-Vieiraet al., 2003). In yeast and Drosophila, the Rad51 paralogsshare the meiotic defects observed in rad51 mutants. Yeast

(e)

(a)

(c)

(f)

(b)

(d)

Figure 5. DAPI staining of wild-type meiotic chromosomes.(a) Prophase I, (b) metaphase I, (c) anaphase I, (d) metaphase II, (e) anaphase IIand (f) telophase II. Arrowheads indicate bivalents (b) or chromosomes (c–e).Scale bars " 10 lm. White dotted lines have been added to clearly indicatethe separate sets of chromosomes.

(e)(e) (f)(f)

(a)(a)

(c)(c)

(b)

(d)(d)

(g)(g)

(i)(i)

(h)(h)

(j)(j)

Figure 6. Meiosis is severely disturbed in atrad51c-1 pollen mother cells.(a) Prophase I, (b, c) metaphase I, (d, e) anaphase I, (f, g) metaphase II, (h, i)anaphase II and (j) telophase II. Arrowheads indicate bridges. Scalebars " 10 lm.



rad55 and rad57 mutants are unable to repair meiotic DSBsand have reduced spore viability and oogenesis and fertilityare severely disturbed in Drosophila spnB (xrcc3) and spnD(rad51c) mutants (Abdu et al., 2003; Game and Mortimer,1974; Ghabrial and Schupbach, 1999; Ghabrial et al., 1998;Schwacha and Kleckner, 1997).

Similarly, Arabidopsis AtRad51 and AtXrcc3 proteins areboth required to achieve meiosis and repair meiotic DSBs(Bleuyard and White, 2004; Li et al., 2004). In contrast toatxrcc3 mutants, the absence of meiotic homologous chro-mosome synapsis in atrad51-1 mutants shows that AtRad51and AtXrcc3 proteins have distinct roles in meiosis, AtRad51acting prior to AtXrcc3. Osakabe et al. (2002) have shownthat AtXrcc3 and AtRad51C can interact together and thuspresumably form a heterodimer in vivo, as is the case invertebrates. In this study, we show that atrad51c-1 mutantplants present meiotic defects similar to those observed inthe atxrcc3 mutant plants (Figure 6), confirming the meioticrole of the Arabidopsis CX3 complex. Mutations in theATSPO11-1 gene, the Arabidopsis SPO11 homolog, dramat-ically reduce meiotic HR and homologous chromosomesynapsis (Grelon et al., 2001). Absence of Spo11 activity inthe atxrcc3 mutant suppresses the chromosome fragmen-tation in half the meiotic cells and delays fragmentation tothe second meiotic division in the other half (Bleuyard et al.,2004), raising the possibility that themeiosis II defects derivefrom unresolved sister chromatid HR events. In addition, Liuet al. (2004) reported that Rad51C plays a major role in HJbranch migration and resolution activities, while Xrcc3 isinvolved in HJ resolution. Taken together, these findingsstrongly suggest that the CX3 complex is involved in theSpo11 meiotic recombination pathway, presumably in theHJ resolution.

Meiotic requirement for the Rad51 paralog proteins

Neither atrad51b-1 nor atxrcc2-1 mutants present visiblemeiotic defects. A trivial explanation for this would be thatputative truncated proteins produced from incompletetranscripts of these alleles are able to carry out the meioticfunctions of the native proteins. However, the atrad51b-1and atxrcc2-1 mutant plants are hypersensitive to DNAcross-linking agents, indicating defects in homologousrecombinational repair of ICLs (Figure 3). Furthermore, thestudies performed to identify interaction domains withinthe Rad51 paralogs have shown that any deletion in eitherthe N-terminal or the C-terminal parts of the proteinseliminate protein–protein interactions (Dosanjh et al., 1998;Kurumizaka et al., 2003; Miller et al., 2004). This finding ledthe authors to suggest that even a very short deletion canseverely disturb the folding of the Rad51 paralogs (Milleret al., 2004). It thus appears very unlikely that putativetruncated proteins produced in either atrad51b-1 oratxrcc2-1 would be functional.

Our finding that the AtRad51B and AtXrcc2 proteins arenot required to achieve meiosis shows that only the CX3complex plays an essential role for the repair of AtSpo11-1induced DSBs. A recent study by Liu et al. (2004) has shownthat the mammalian Rad51C and Xrcc3 proteins are bothinvolved in HJ resolution, while the other Rad51 paralogsare implicated in branch migration processes. These resultsstrongly support the idea that the CX3 and BCDX2 (or at leastthe Rad51B, Rad51C and Xrcc2 proteins) complexes havedistinct roles in HR mechanisms and hence in meioticrecombination.

In the absence of the AtXrcc3 protein, meiosis is severelydisturbed (Bleuyard andWhite, 2004), indicating that the CX3complex has an essential function during meiosis and thatthis function cannot be complemented by the BCDX2 com-plex. In addition, one might expect that mutations in theATRAD51C gene lead to more critical defects, due to thedisruption of both CX3 and BCDX2 complexes. However,atrad51c-1 and atxrcc3 mutants present very similar defects(this study; Bleuyard and White, 2004), supporting theexistence of an essential role for the CX3 complex duringmeiosis, while the BCDX2 complex is dispensable. At thispoint we cannot however exclude the possibility that theBCDX2 complex plays a non-essential role in meiotic recom-bination processes in contrast to the essential role of the CX3complex (resolvase activity?), absence of which leads tochromosome fragmentation in thefirstmeiotic prophase.Wenote that, although very probable, we cannot be certain thattheArabidopsisAtRad51B,AtRad51C,AtRad51DandAtXrcc2proteins form a BCDX2 complex in vivo, as this has not yetbeen formally tested. Our results show the absence ofessentialmeiotic roles for theAtRad51BandAtXrcc2proteinsin Arabidopsis, but that this conclusion also applies to theBCDX2 complex must remain tentative until formal demon-stration of the existence of the complex in this plant.

In vertebrates, the embryonic lethality of knockoutanimals has greatly complicated studies of the meioticroles of Rad51-like proteins (Deans et al., 2000; Pittmanand Schimenti, 2000; Shu et al., 1999; Tsuzuki et al., 1996).In contrast to other model organisms, Arabidopsis carriesthe same range of Rad51-like proteins as vertebrates andmutants defective for Rad51 or any of the Rad51 paralogsare viable (this study; Bleuyard and White, 2004; Li et al.,2004). With the recent availability of public, sequence-tagged mutant collections, Arabidopsis thus shows greatpromise as a model to study the meiotic functions ofproteins involved in recombination.

Experimental procedures

Phylogenetic analysis

Sequence alignments were carried out using the ClustalX softwarepackage (Version 1.83, Thompson et al., 1997). Evolutionary



distances were calculated using the Henifoff/Tillier PMB (ProbabilityMatrix from Blocks, Veerassamy et al., 2003) distancemethod of theProtdist program (PHYLIP package version 3.6, Felsenstein, 1989). Thecoefficient of variation of the c-distribution (to incorporate rateheterogeneity)was obtained bypre-analyzing the datawith the Tree-Puzzle program (Version 5.0, Strimmer and von Haeseler, 1997). Thephylogenetic tree was inferred using the unweighted pair groupmethod with arithmetic mean method in the neighbor program(PHYLIP package version 3.6, Felsenstein, 1989). The tree was dis-played using TreeView program (version 1.6.6). Consensus treeswere inferred using the Consense program (PHYLIP package version3.6, Felsenstein, 1989) and the significance of the various phylo-genetic lineageswasassessedbybootstrap analyses (Hedges, 1992).

Plant material, growth conditions and mutant screening

All Arabidopsis plants used in this work were of ecotype Columbia(Col0). A. thaliana seeds were sown directly into damp compost orsolid germination medium and under white light (16 h light/8 hdark) as previously described by Gallego et al. (2001).

The atrad51b-1 (Salk_024755), atrad51c-1 (Salk_021960) andatxrcc2-1 (Salk_029106) T-DNA insertion lines were found in thepublic T-DNA Express database established by the Salk InstituteGenomic Analysis Laboratory accessible from the SIGnAL websiteat http://signal.salk.edu (Alonso et al., 2003).

Plants heterozygous and/or homozygous for the atrad51b-1,atrad51c-1 or atxrcc2-1 T-DNA insertion loci were identified by aPCR genotyping assay. The following primer combinations wereused to amplify the different loci: the wild type ATRAD51B locus,o519 (5¢-GAGTTAGTTGGTCCTCCTGG-3¢) and o520 (5¢-AAA-TTCAGCAAGCGATCTGG-3¢); the atrad51b-1 mutant locus, o519and o405 (5¢-TGGTTCACGTAGTGGGCCATCG-3¢); the wild typeATRAD51C locus, o527 (5¢-TTTTGTGACTAAACAAAGGAGC-3¢)and o528 (5¢-ACCTCCACTTAAGCTAGTCAAGG-3¢); the atrad51c-1mutant locus, o527 and o405; the wild type ATXRCC2 locus, o523(5¢-TAGTCCAATGTAACTTTCGCAG-3¢) and o524 (5¢-GTCACGAGA-CAATGACAATACC-3¢); the atxrcc2-1 mutant locus, o523 and o405.atrad51c-1 mutant plants identification was confirmed based ontheir sterility phenotype.

Sequencing of T-DNA insertion sites

The following primer combinations were used to amplify DNAflanking the T-DNA: atrad51b-1 LB1 left border, o519 and o405;atrad51b-1 LB2 left border, o520 and o405; atrad51c-1 LB1 left bor-der, o527 and o405; atrad51c-1 LB2 left border, o528 and o405; theatxrcc2-1 LB1 left border, o523 and o405; the atxrcc2-1 LB2 leftborder, o524 and o405.

The PCR products were then purified on a QIAquick column(Qiagen, Courtaboeuf, France) and directly sequenced. Sequencereaction were performed using one of the primers used foramplification and the CEQ DTCS Quick Start Kit (Beckman Coulter,Fullerton, CA, USA), and analyzed on a CEQ 2000 DNA AnalysisSystem (Beckman Coulter).

Semiquantitative RT-PCR

For semiquantitative RT-PCR, total RNAs extracted from flowerbuds were treated with RNase-free DNase I (Roche, Meylan,France). One microgram of DNA-free total RNA was reversetranscribed in 20 ll of reaction mixture containing 50 units ofExpand Reverse Transcriptase (Roche), 1X random hexanucleotide

mix (Roche), 1 mM of each deoxyribonucleotide triphosphate, and20 units of RNasin ribonuclease inhibitor (Promega, Charbon-nieres, France). PCR was performed in 25 ll reaction mixturescontaining 2 ll of RT reaction mixture, 1 unit of HotStarTaq DNApolymerase (Qiagen), 2.5 mM MgCl2, 100 lM of each deoxyribo-nucleotide triphosphate, and 0.4 lM of gene-specific primers.

The gene-specific primers were: o548 (5¢-TTTCCAGTAGCTTATG-GAGG-3¢) and o549 (5¢-ATATGCCAACCCAACTGAGG-3¢), or o546(5¢-AGTGAAGCTACTTCTCCACC-3¢) and o547 (5¢-CCGGAAAGCTT-TCCAGTCCC-3¢) for ATRAD51B; o453 (5¢-CTTGATAACATTTT-GGGCGG-3¢) and o454 (5¢-CAAGATGATTGACCAATGCG-3¢), oro450 (5¢-ATGATTTCATTTGGGCGGCG-3¢) and o554 (5¢-TAATAC-GCGGCAAAGACTCC-3¢) for ATRAD51C; o552 (5¢-GCATTGGTGCTT-TTCACTGG-3¢) and o553 (5¢-ATTCACGAAATGGAGGTTGC-3¢), oro550 (5¢-GAAGCAGATGTTATCAAGGG-3¢) and o551 (5¢-CCATGCTC-CATTTCCTAACC-3¢) for ATXRCC2. The initial denaturation wasperformed at 95!C for 15 min, then amplification was performed for45 cycles with a denaturation time of 30 secec at 94!C, followed byannealing for 30 sec at 58!C and extension for 45 sec at 72!C. TheAPT1 (adenine phosphorybosyl transferase) transcript has beenused as a control for reverse transcription (Moffatt et al., 1994). Thegene-specific primers were apt1 (5¢-TCCCAGAATCGCTAAGATTGC-3¢) and apt2 (5¢-CCTTTCCCTTAA-GCTCTG-3¢). The initial denatura-tion was performed at 95!C for 15 min, then amplification wasperformed for 35 cycles with a denaturation time of 30 sec at 94!C,followed by annealing for 30 sec at 52!C and extension for 45 sec at72!C.

Mitomycin C and c-irradiation assays

Col0, atrad51b-1, atrad51c-1 and atxrcc2-1 seeds were surface-sterilized with 7% calcium hypochlorite solution (w/v).

For MMC assays, seeds were sown on plates containingfresh solid germination medium with different concentrations ofMMC (Sigma no. M-0503, Sigma, Lyon, France). The plates werethen incubated for 2 weeks (23!C, 16 h light), and resistance orsensitivity was scored by the number of true leaves (excludingthe cotyledons) per plant.

For c-irradiation, surface-sterilized seeds were kept in sterilewater at 4!C for approximately 24 h. Then seeds were exposed to50, 100 or 200 Gy (9.12 Gy min)1) from a 137Cs source (CIS BioInternational, Gif sur Yvette, France) and sown on plates containingfresh solid germinationmedium. The plates were then incubated for2 weeks (23!C, 16 h light), and resistance or sensitivity was scoredas for the MMS treatment.

Light and fluorescence microscopy

Cytological observations of Alexander-stained anthers, embryo sacdevelopment and meiotic chromosomes were conducted as previ-ously described (Bleuyard and White, 2004). Images were capturedon a Zeiss Axioplan 2 Imaging microscope with a Zeiss AxiocamHRc video camera (Zeiss, Le Pecq, France) and enhanced usingAdobe Photoshop 6 software.

Acknowledgements

We thank members of BIOMOVE for their help and discussions andthe Salk Institute Genomic Analysis Laboratory for providing thesequence-indexed Arabidopsis T-DNA insertion mutants. We alsothank Hong Ma and Bernd Reiss for communicating their data to usbefore publication.



This work was partly financed by a European Union researchgrant (QLG2-CT-2001-01397), the Centre National de la RechercheScientifique and the Universite Blaise Pascal.

References

Abdu, U., Gonzalez-Reyes, A., Ghabrial, A. and Schupbach, T. (2003)The Drosophila spn-D gene encodes a RAD51C-like protein that isrequired exclusively during meiosis. Genetics, 165, 197–204.

Aboussekhra, A., Chanet, R., Adjiri, A. and Fabre, F. (1992) Semi-dominant suppressors of Srs2 helicase mutations of Saccharo-myces cerevisiae map in the RAD51 gene, whose sequencepredicts a protein with similarities to prokaryotic RecA proteins.Mol. Cell Biol. 12, 3224–3234.

Albala, J.S., Thelen, M.P., Prange, C., Fan, W., Christensen, M.,Thompson, L.H. and Lennon, G.G. (1997) Identification of a novelhuman RAD51 homolog, RAD51B. Genomics, 46, 476–479.

Alexander, M.P. (1969) Differential staining of aborted and non-aborted pollen. Stain Technol. 44, 117–122.

Alonso, J.M., Stepanova, A.N., Leisse, T.J. et al. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science,301, 653–657.

Bishop, D.K., Ear, U., Bhattacharyya, A., Calderone, C., Beckett, M.,Weichselbaum, R.R. and Shinohara, A. (1998) Xrcc3 is requiredfor assembly of Rad51 complexes in vivo. J. Biol. Chem. 273,21482–21488.

Bleuyard, J.Y. and White, C.I. (2004) The Arabidopsis homologue ofXrcc3 plays an essential role in meiosis. EMBO J. 23, 439–449.

Bleuyard, J.Y., Gallego, M.E. and White, C.I. (2004) The atspo11-1mutation rescues atxrcc3 meiotic chromosome fragmentation.Plant Mol. Biol. in press.

Brenneman, M.A., Weiss, A.E., Nickoloff, J.A. and Chen, D.J. (2000)XRCC3 is required for efficient repair of chromosome breaks byhomologous recombination. Mutat. Res. 459, 89–97.

Brenneman, M.A., Wagener, B.M., Miller, C.A., Allen, C. and Nick-oloff, J.A. (2002) XRCC3 controls the fidelity of homologousrecombination: roles for XRCC3 in late stages of recombination.Mol. Cell 10, 387–395.

Cao, L., Alani, E. and Kleckner, N. (1990) A pathway for generationand processing of double-strand breaks during meiotic recom-bination in S. cerevisiae. Cell, 61, 1089–1101.

Cartwright, R., Tambini, C.E., Simpson, P.J. and Thacker, J. (1998a)The XRCC2 DNA repair gene from human and mouse encodes anovel member of the recA/RAD51 family. Nucleic Acids Res. 26,3084–3089.

Cartwright, R., Dunn, A.M., Simpson, P.J., Tambini, C.E. andThacker, J. (1998b) Isolation of novel human and mouse genes ofthe recA/RAD51 recombination-repair gene family. Nucleic AcidsRes. 26, 1653–1659.

Cui, X., Brenneman, M., Meyne, J., Oshimura, M., Goodwin, E.H.and Chen, D.J. (1999) The XRCC2 and XRCC3 repair genes arerequired for chromosome stability in mammalian cells. Mutat.Res. 434, 75–88.

Deans, B., Griffin, C.S., Maconochie, M. and Thacker, J. (2000) Xrcc2is required for genetic stability, embryonic neurogenesis andviability in mice. EMBO J. 19, 6675–6685.

Deans, B., Griffin, C.S., O’Regan, P., Jasin, M. and Thacker, J. (2003)Homologous recombination deficiency leads to profound geneticinstability in cells derived from Xrcc2-knockout mice. Cancer Res.63, 8181–8187.

Dosanjh, M.K., Collins, D.W., Fan, W., Lennon, G.G., Albala, J.S.,Shen, Z. and Schild, D. (1998) Isolation and characterization ofRAD51C, a new human member of the RAD51 family of relatedgenes. Nucleic Acids Res. 26, 1179–1184.

Doutriaux, M.P., Couteau, F., Bergounioux, C. and White, C. (1998)Isolation and characterisation of the RAD51 and DMC1 homologsfrom Arabidopsis thaliana. Mol. Gen. Genet. 257, 283–291.

Dudas, A. and Chovanec, M. (2004) DNA double-strand break repairby homologous recombination. Mutat. Res. 566, 131–167.

Felsenstein, J. (1989) PHYLIP (phylogeny inference package). Cla-distics, 5, 164–166.

French, C.A., Masson, J.Y., Griffin, C.S., O’Regan, P., West, S.C. andThacker, J. (2002) Role of mammalian RAD51L2 (RAD51C) inrecombination and genetic stability. J. Biol. Chem. 277, 19322–19330.

Friesner, J. and Britt, A.B. (2003) Ku80- and DNA ligase IV-deficientplants are sensitive to ionizing radiation and defective in T-DNAintegration. Plant J. 34, 427–440.

Gallego, M.E., Jeanneau, M., Granier, F., Bouchez, D., Bechtold, N.and White, C.I. (2001) Disruption of the Arabidopsis RAD50 geneleads to plant sterility and MMS sensitivity. Plant J. 25, 31–41.

Game, J.C. and Mortimer, R.K. (1974) A genetic study of X-raysensitive mutants in yeast. Mutat. Res. 24, 281–292.

Garcia, V., Bruchet, H., Camescasse, D., Granier, F., Bouchez, D. andTissier, A. (2003) AtATM is essential for meiosis and the somaticresponse to DNA damage in plants. Plant Cell, 15, 119–132.

Ghabrial, A. and Schupbach, T. (1999) Activation of a meioticcheckpoint regulates translation of Gurken during Drosophilaoogenesis. Nat. Cell Biol. 1, 354–357.

Ghabrial, A., Ray, R.P. and Schupbach, T. (1998) okra and spindle-Bencode components of the RAD52 DNA repair pathway and affectmeiosis and patterning in Drosophila oogenesis. Genes Dev. 12,2711–2723.

Godthelp, B.C., Wiegant, W.W., van Duijn-Goedhart, A., Scharer,O.D., van Buul, P.P., Kanaar, R. and Zdzienicka, M.Z. (2002)Mammalian Rad51C contributes to DNA cross-link resistance,sister chromatid cohesion and genomic stability. Nucleic AcidsRes. 30, 2172–2182.

Grelon, M., Vezon, D., Gendrot, G. and Pelletier, G. (2001) AtSPO11-1 is necessary for efficient meiotic recombination in plants. EMBOJ. 20, 589–600.

Hedges, S.B. (1992) The number of replications needed for accurateestimation of the bootstrap P value in phylogenetic studies. Mol.Biol. Evol. 9, 366–369.

Higgins, C.F., Hiles, I.D., Whalley, K. and Jamieson, D.J. (1985)Nucleotide binding by membrane components of bacterial peri-plasmic binding protein-dependent transport systems. EMBO J.4, 1033–1039.

Johnson, R.D., Liu, N. and Jasin, M. (1999) Mammalian XRCC2promotes the repair of DNA double-strand breaks by homologousrecombination. Nature, 401, 397–399.

Klimyuk, V.I. and Jones, J.D. (1997) AtDMC1, the Arabidopsishomologue of the yeast DMC1 gene: characterization, transpo-son-induced allelic variation and meiosis-associated expression.Plant J. 11, 1–14.

Kurumizaka, H., Ikawa, S., Nakada, M., Eda, K., Kagawa, W., Takata,M., Takeda, S., Yokoyama, S. and Shibata, T. (2001) Homologous-pairing activity of the human DNA-repair proteins Xrcc3.Rad51C.Proc. Natl Acad. Sci. USA, 98, 5538–5543.

Kurumizaka, H., Ikawa, S., Nakada, M., Enomoto, R., Kagawa, W.,Kinebuchi, T., Yamazoe, M., Yokoyama, S. and Shibata, T. (2002)Homologous pairing and ring and filament structure formationactivities of the human Xrcc2*Rad51D complex. J. Biol. Chem.277, 14315–14320.

Kurumizaka, H., Enomoto, R., Nakada, M., Eda, K., Yokoyama, S.and Shibata, T. (2003) Region and amino acid residues requiredfor Rad51C binding in the human Xrcc3 protein. Nucleic AcidsRes. 31, 4041–4050.



Lees-Miller, S.P. and Meek, K. (2003) Repair of DNA double strandbreaksbynon-homologousend joining.Biochimie,85, 1161–1173.

Li, W., Chen, C., Markmann-Mulisch, U., Timofejeva, L., Schmelzer,E., Ma, H. and Reiss, B. (2004) The Arabidopsis AtRAD51 gene isdispensable for vegetative development but required for meiosis.Proc. Natl Acad. Sci. USA, 101, 10596–10601.

Lio, Y.C., Mazin, A.V., Kowalczykowski, S.C. and Chen, D.J. (2003)Complex formation by the human Rad51B and Rad51C DNA re-pair proteins and their activities in vitro. J. Biol. Chem. 278, 2469–2478.

Liu, N. (2002) XRCC2 is required for the formation of Rad51 fociinduced by ionizing radiation and DNA cross-linking agent Mito-mycin C. J. Biomed. Biotechnol. 2, 106–113.

Liu, N., Lamerdin, J.E., Tebbs, R.S. et al. (1998) XRCC2 and XRCC3,new human Rad51-family members, promote chromosome sta-bility and protect against DNA cross-links and other damages.Mol. Cell, 1, 783–793.

Liu, N., Schild, D., Thelen, M.P. and Thompson, L.H. (2002)Involvement of Rad51C in two distinct protein complexes ofRad51 paralogs in human cells. Nucleic Acids Res. 30, 1009–1015.

Liu, Y., Masson, J.Y., Shah, R., O’Regan, P. and West, S.C. (2004)RAD51C is required for Holliday junction processing in mamma-lian cells. Science, 303, 243–246.

Masson, J.Y., Tarsounas, M.C., Stasiak, A.Z., Stasiak, A., Shah, R.,McIlwraith, M.J., Benson, F.E. andWest, S.C. (2001) Identificationand purification of two distinct complexes containing the fiveRAD51 paralogs. Genes Dev. 15, 3296–3307.

Miller, K.A., Yoshikawa, D.M., McConnell, I.R., Clark, R., Schild, D.and Albala, J.S. (2002) RAD51C interacts with RAD51B and iscentral to a larger protein complex in vivo exclusive of RAD51. J.Biol. Chem. 277, 8406–8411.

Miller, K.A., Sawicka, D., Barsky, D. and Albala, J.S. (2004) Domainmapping of the Rad51 paralog protein complexes. Nucleic AcidsRes. 32, 169–178.

Moffatt, B.A., McWhinnie, E.A., Agarwal, S.K. and Schaff, D.A.(1994) The adenine phosphoribosyltransferase-encoding gene ofArabidopsis thaliana. Gene, 143, 211–216.

Mohindra, A., Bolderson, E., Stone, J., Wells, M., Helleday, T. andMeuth, M. (2004) A tumour-derived mutant allele of XRCC2preferentially suppresses homologous recombination at DNAreplication forks. Hum. Mol. Genet. 13, 203–212.

O’Regan, P., Wilson, C., Townsend, S. and Thacker, J. (2001) XRCC2is a nuclear RAD51-like protein required for damage-dependentRAD51 focus formation without the need for ATP binding. J. Biol.Chem. 276, 22148–22153.

Osakabe, K., Yoshioka, T., Ichikawa, H. and Toki, S. (2002)Molecularcloning and characterization of RAD51-like genes from Arabid-opsis thaliana. Plant Mol. Biol. 50, 71–81.

Pierce, A.J., Johnson, R.D., Thompson, L.H. and Jasin, M. (1999)XRCC3 promotes homology-directed repair of DNA damage inmammalian cells. Genes Dev. 13, 2633–2638.

Pittman, D.L. and Schimenti, J.C. (2000) Midgestation lethality inmice deficient for the RecA-related gene, Rad51d/Rad51l3. Gen-esis, 26, 167–173.

Pittman, D.L., Weinberg, L.R. and Schimenti, J.C. (1998) Identifica-tion, characterization, and genetic mapping of Rad51d, a newmouse and human RAD51/RecA-related gene.Genomics, 49, 103–111.

Rice, M.C., Smith, S.T., Bullrich, F., Havre, P. and Kmiec, E.B. (1997)Isolation of human and mouse genes based on homology toREC2, a recombinational repair gene from the fungus Ustilagomaydis. Proc. Natl Acad. Sci. USA, 94, 7417–7422.

Rinaldo, C., Bazzicalupo, P., Ederle, S., Hilliard, M. and La Volpe, A.(2002) Roles for Caenorhabditis elegans rad-51 in meiosis and in

resistance to ionizing radiation during development. Genetics,160, 471–479.

Sato, S., Hotta, Y. and Tabata, S. (1995) Structural analysis of arecA-like gene in the genome ofArabidopsis thaliana.DNARes. 2,89–93.

Schild, D., Lio, Y.C., Collins, D.W., Tsomondo, T. and Chen, D.J.(2000) Evidence for simultaneous protein interactions betweenhuman Rad51 paralogs. J. Biol. Chem. 275, 16443–16449.

Schwacha, A. and Kleckner, N. (1997) Interhomolog bias duringmeiotic recombination: meiotic functions promote a highly dif-ferentiated interhomolog-only pathway. Cell, 90, 1123–1135.

Shinohara, A., Ogawa, H. and Ogawa, T. (1992) Rad51 protein in-volved in repair and recombination in S. cerevisiae is a RecA-likeprotein. Cell, 69, 457–470.

Shu, Z., Smith, S., Wang, L., Rice, M.C. and Kmiec, E.B. (1999) Dis-ruption of muREC2/RAD51L1 in mice results in early embryoniclethality which can be partially rescued in a p53()/)) background.Mol. Cell Biol. 19, 8686–8693.

Sigurdsson, S., Van Komen, S., Bussen, W., Schild, D., Albala, J.S.and Sung, P. (2001) Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange.Genes Dev. 15, 3308–3318.

Sonoda, E., Sasaki, M.S., Buerstedde, J.M., Bezzubova, O., Shino-hara, A., Ogawa, H., Takata, M., Yamaguchi-Iwai, Y. and Takeda,S. (1998) Rad51-deficient vertebrate cells accumulate chromoso-mal breaks prior to cell death. EMBO J. 17, 598–608.

Staeva-Vieira, E., Yoo, S. and Lehmann, R. (2003) An essential roleof DmRad51/SpnA in DNA repair and meiotic checkpoint control.EMBO J. 22, 5863–5874.

Strimmer, K. and von Haeseler, A. (1997) Likelihood-mapping: asimple method to visualize phylogenetic content of a sequencealignment. Proc. Natl Acad. Sci. USA, 94, 6815–6819.

Sung, P. (1997) Yeast Rad55 and Rad57 proteins form a heterodimerthat functions with replication protein A to promote DNA strandexchange by Rad51 recombinase. Genes Dev. 11, 1111–1121.

Symington, L.S. and Holloman, W.K. (2004) Molecular biology. NewYear’s resolution – resolving resolvases. Science, 303, 184–185.

Takata, M., Sasaki, M.S., Sonoda, E., Fukushima, T., Morrison, C.,Albala, J.S., Swagemakers, S.M., Kanaar, R., Thompson, L.H. andTakeda, S. (2000) The Rad51 paralog Rad51B promotes homol-ogous recombinational repair. Mol. Cell Biol. 20, 6476–6482.

Takata, M., Sasaki, M.S., Tachiiri, S., Fukushima, T., Sonoda, E.,Schild, D., Thompson, L.H. and Takeda, S. (2001) Chromosomeinstability and defective recombinational repair in knockout mu-tants of the five Rad51 paralogs. Mol. Cell Biol. 21, 2858–2866.

Tarsounas, M., Davies, A.A. and West, S.C. (2004) RAD51 localiza-tion and activation following DNA damage. Philos. Trans. R. Soc.Lond. B Biol. Sci. 359, 87–93.

Tebbs, R.S., Zhao, Y., Tucker, J.D., Scheerer, J.B., Siciliano, M.J.,Hwang, M., Liu, N., Legerski, R.J. and Thompson, L.H. (1995)Correction of chromosomal instability and sensitivity to diversemutagens by a cloned cDNA of the XRCC3 DNA repair gene. Proc.Natl Acad. Sci. USA, 92, 6354–6358.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. andHiggins, D.G. (1997) The CLUSTAL_X windows interface: flexiblestrategies for multiple sequence alignment aided by qualityanalysis tools. Nucleic Acids Res. 25, 4876–4882.

Tsuzuki, T., Fujii, Y., Sakumi, K., Tominaga, Y., Nakao, K., Sekiguchi,M., Matsushiro, A., Yoshimura, Y. and Morita, T. (1996) Targeteddisruption of the Rad51 gene leads to lethality in embryonic mice.Proc. Natl Acad. Sci. USA, 93, 6236–6240.

Urban, C., Smith, K.N. and Beier, H. (1996) Nucleotide sequences ofnuclear tRNA(Cys) genes from Nicotiana and Arabidopsis andexpression in HeLa cell extract. Plant Mol. Biol. 32, 549–552.



Veerassamy, S., Smith, A. and Tillier, E.R. (2003) A transitionprobability model for amino acid substitutions from blocks. J.Comput. Biol. 10, 997–1010.

Walker, J.E., Saraste, M., Runswick, M.J. and Gay, N.J. (1982) Dis-tantly related sequences in the alpha- and beta-subunits of ATPsynthase, myosin, kinases and other ATP-requiring enzymes anda common nucleotide binding fold. EMBO J. 1, 945–951.

Wiese, C., Collins, D.W., Albala, J.S., Thompson, L.H., Kronenberg,A. and Schild, D. (2002) Interactions involving the Rad51 paralogsRad51C and XRCC3 in human cells. Nucleic Acids Res. 30, 1001–1008.

Yokoyama, H., Kurumizaka, H., Ikawa, S., Yokoyama, S. and Shi-bata, T. (2003) Holliday junction binding activity of the humanRad51B protein. J. Biol. Chem. 278, 2767–2772.

Yokoyama, H., Sarai, N., Kagawa, W., Enomoto, R., Shibata, T.,Kurumizaka, H. and Yokoyama, S. (2004) Preferential binding tobranched DNA strands and strand-annealing activity of thehuman Rad51B, Rad51C, Rad51D and Xrcc2 protein complex.Nucleic Acids Res. 32, 2556–2565.



Documents

Differing requirements for the Arabidopsis Rad51 paralogs in meiosis and DNA repair